The insulin-like growth factor (IGF)-independent effects of insulin-like growth factor binding protein-3 (IGFBP-3) to effect cellular apoptosis have now been described in various cellular systems. IGFBP-3 mediates transforming growth factor-beta-induced apoptosis. Other growth-inhibitory and apoptosis-inducing agents such as tumor necrosis factor-alpha (TNF-alpha) and the tumor suppressor gene p53 also induce IGFBP-3. In this report, we demonstrate the role of IGFBP-3 as a mediator of apoptosis induced by TNF-alpha and elucidate the process involved in its signaling mechanism. Treatment of PC-3 cells with TNF-alpha resulted in the induction of IGFBP-3 expression in a dose- and time-dependent fashion and also induced apoptosis. TNF-alpha-induced apoptosis was prevented by cotreatment with IGFBP-3 neutralizing antibodies or IGFBP-3-specific antisense thiolated oligonucleotides. Both IGFBP-3 and TNF-alpha treatment increased the levels of the inactive, serine phosphorylated form of the survival protein Bcl-2. The effect of TNF-alpha on Bcl-2 serine phosphorylation was blocked by IGFBP-3 antisense oligomers. These findings confirm that IGFBP-3 is essential for TNF-alpha-induced apoptosis in PC-3 cells and that this IGFBP-3 effect includes the inactivation of Bcl-2 through serine phosphorylation.